Lentiviral vectors (LVs) and other viral vectors are an attractive tool for gene therapy (Thomas et al., 2003). LVs can transduce a broad range of tissues, including nondividing cells such as hepatocytes, neurons and hematopoietic stem cells. Moreover, LVs integrate into target cell genomes and provide long-term transgene expression.
Although LVs can provide efficient and stable gene transfer, targeting expression to, or de-targeting expression from, a specific cell type remains difficult. This problem is particularly relevant following in vivo vector administration in which transgene expression may only be desired in a specific cell population, such as tumor cells or hepatocytes, but a broad spectrum of cell types are transduced. De-targeting expression is also important when progenitor or stem cells are transduced, but it is necessary to have transgene expression restricted to only one particular lineage of the differentiated population. To date, most efforts to address this problem have relied on either targeting the vector envelope or engineering tissue-specific promoters. There are, however, limitations with both these methods.
Targeted envelopes can reduce the vector titer and result in decreased vector infectivity (Sandrin et al., 2003). Tissue-specific promoters, which are constructed based on, but not identical to, naturally occurring promoter/enhancer elements, are often weakly expressed in target tissues compared to ubiquitously expressed promoters. In addition, these tissue-specific promoters do not always achieve absolute cell specificity (Follenzi et al., 2002). Transgene expression in non-target cells can occur for a variety of reasons, including ‘leaky’ promoter activity and promoter/enhancer trapping (De Palma et al., 2005). The trapping phenomenon comes about because the vector preferentially integrates at sites of active transcription, which can, in turn, drive transgene transcription independent of the vector's promoter.
In order to circumvent these problems and create a vector that can maintain high infectivity and robust expression, while enabling tight restriction of transgene expression from particular cell types, we developed a vector that is regulated by endogenously expressed microRNA (miRNA).
WO03/020931 describes an reporter system assay system displaying miRNA provides a method for measuring knockdown of a readily assayed gene. The system is used to determine if siRNAs and chimeric RNAs can decrease expression of the readily assayed luciferase gene.
US Patent Application 20050266552 describes the construction of a reporter construct suitable for introduction into mammalian cells to create cell lines that can be used for identification of genes involved in miRNA translational repression pathways and/or chemical modulators of such pathways.
Mansfield J H et al (2004) Nat Genet 36(10):1079-83 Epub, erratum in Nat Genet (2004) 36(11):1238; and Brennecke J et al (2005) PloS Biol 3(3):e85 both describe plasmids containing a reporter gene with miRNA target sequences. In both reports, the constructs were designed to monitor expression of endogenous miRNAs and not for the purpose of regulating a transgene and/or restricting expression to particular cell types.
An important feature of our invention that should be highlighted is that we describe how vectors can be designed to be regulated by endogenous miRNAs for controlling transgene expression to achieve specific expression profiles of the vector. Although reports already exist, which demonstrate that miRNA target sequences can be included in a reporter construct (a plasmid expressing a marker gene such as luciferase) to track expression of a miRNA, they do not describe exploiting miRNAs specifically for vector regulation. They particularly do not describe the use of the vectors of the present invention for gene therapy approaches to prevent immune mediated rejection of a transgene of interest or manufacturing approaches to increase titer of viral particles that express toxic genes which are normally toxic to the cell in which the viral particle is produced.